JPH0843375A - Hair tonic evaluating method - Google Patents

Abstract

PURPOSE:To evaluate properly the actual hair growth promoting effect by measuring the activeness of the cornification enzyme contained in a trichocyte or a trichocyte-originated cultured cell of a human being or any experimental animal to which a hair tonic was given. CONSTITUTION:A cornification enzyme is used to measure the enzyme activeness for evaluation of the hair growth promoting effect of a hair tonic, wherein any enzyme to have commitment with cornification may be used, but a favorable one is sulfhydryl oxidase (SH oxidase) having the effect of promoting synthetization of cystine. To measure the activeness of the cornification enzyme in a haired skin, a hair is drawn off and its root is sampled, to which a buffer liquid of sodium phosphate is added to generate homonization, and the obtained homogenate is subjected to a centrifugal separating process and the supernatent thereof is used as a specimen for measurement of the degree of light absorptiveness. From the obtained measurements, the value of activenss of the enzyme is determined and is divided by the protein content of the supernatent, and the resultant is used as the index for evaluation. This permits knowing the condition of the hair, and the hair growth promoting effect can be evaluated properly.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for evaluating a hair nourishing agent, and more particularly to a method for evaluating a hair nourishing agent, which measures a keratinizing enzyme activity value and evaluates the hair nourishing effect using this as an index.

[0002]

2. Description of the Related Art Beautiful and rich hair has been a longing of humanity since ancient times. However, there are not a few people who lose their hair as they grow older or due to the constitution they were born with, as they age or become younger. And for those who have lost their hair, it is often a psychological burden.

Under these circumstances, various hair restorers and hair restorers have been developed so far. However, most of the conventional hair nourishing agents and hair nourishing agents have used the hair growth promoting action as an index in their development.
At present, these hair nourishing agents and hair nourishing agents have not been checked for effects on hair quality improvement, hair loss improvement, and the like.

Therefore, when a hair nourishing agent or a hair nourishing agent whose hair nourishing effect is highly evaluated is used only by checking the above hair nourishing effect, the hair is promoted, but the hair quality is always hard and thick. It is not improved to hair, and the hair that has grown is thin, soft, easy to cut and easy to pull out, often lacks firmness and volume, and the actual hair nourishing effect is not sufficient There were many

That is, the conventional evaluation of the hair nourishing effect, that is, the evaluation performed by checking only the hair growth promoting effect is insufficient in content, but an effective hair nourishing agent and hair nourishing agent cannot be developed. It can be said that

Therefore, it has been desired to develop a method for evaluating a hair nourishing agent that can appropriately evaluate the actual hair nourishing effect.

[0007]

The present invention has been made from the above viewpoint, and an object of the present invention is to provide a method for evaluating a hair nourishing agent that can appropriately evaluate the actual hair nourishing effect.

[0008]

In order to solve the above problems, the present inventors first examined the actual hair-growth effect and its evaluation method, and as a result, the hair-growth effect actually required is I think that it is the effect of giving the hair a volume feeling, and in order to evaluate the hair nourishing effect that gives the hair a volume feeling, in addition to the evaluation of the hair growth promoting action, make the hair thick, strong and firm, We thought that it would be possible to evaluate the actual hair nourishing effect by evaluating the hair quality-improving effect that gives the body elasticity.

Therefore, as a result of various studies on means for evaluating the hair quality improving effect, a correlation was found between the activity promoting action of keratinizing enzyme and the hair quality improving action, and further, hair follicles of experimental animals or humans to which the hair nourishing agent was administered. Further, the inventors have found that the actual hair-feeding effect can be appropriately evaluated by measuring the activity of keratinizing enzyme in cultured cells derived from hair follicles and evaluating the hair-feeding effect using this as an index.

That is, the present invention is characterized in that the hair-growth effect of said hair-growth agent is evaluated by measuring the activity of keratinizing enzyme in hair follicles or cultured cells derived from hair follicles of experimental animals or humans to which the hair-growth agent is administered. Is a method for evaluating a hair nourishing agent.

Here, the method of evaluating the hair nourishing agent of the present invention is not limited to the hair nourishing agent, but may be a drug containing a hair nourishing agent, a quasi drug,
It is also applied to cosmetics and the like, and the "hair restorer" used below is a generic term for these.

The present invention will be described in detail below. In the method for evaluating a hair nourishing agent of the present invention, the activity of keratinizing enzyme in a hair follicle or a cultured cell derived from a hair follicle of an experimental animal or human to which the hair nourishing agent is administered is measured.

The keratin enzyme whose enzyme activity is measured for the evaluation of the hair nourishing agent of the present invention is not particularly limited as long as it is an enzyme involved in keratinization, but sulfhydryl oxidase (SH oxidase), transglutaminase (T glutaminase). ) And the like, and among these, SH oxidase is preferably used in the present invention.

Keratin enzymes are enzymes involved in the synthesis of keratin, and there are two types of keratin, soft keratin having a low cystine content and hard keratin having a high cystine content, and hair is hard. It is known to consist of keratin, and since the synthesis of cystine is promoted by SH oxidase, SH oxidase is preferable among keratinizing enzymes for use in evaluating hair-growth effect.

In the method for evaluating a hair nourishing agent of the present invention, the sample used for measuring the keratinizing enzyme activity is a hair follicle of an experimental animal or human or a cultured cell derived from a hair follicle. Here, the experimental animals used in the evaluation method of the present invention are not particularly limited, and ordinary experimental animals such as mice, rats, guinea pigs, rabbits and pigs can be used.

Regarding the hair follicle, it is not always necessary to use the entire hair follicle, and it may be a part of the hair follicle such as a hair bulb portion such as hair mother cells or a hair papilla portion. As a method for obtaining such a hair follicle or a part of the hair follicle from the above-mentioned laboratory animal or human, a method of collecting body hair or hair in a form containing the hair follicle or a part of the hair follicle and obtaining from the hair root Is mentioned.

Further, the sample for measuring the activity of keratinase is
It may contain cells other than hair follicles, and even if skin tissues other than hair follicles are attached to the hair root obtained as described above, it is possible to use them as they are. Further, as for the experimental animal, instead of collecting body hair, for example, a method of collecting dehaired skin or the like is used to prepare the entire skin tissue including hair follicles and directly use the same in the evaluation method of the present invention. It is also possible.

Instead of using each of the above samples, hair follicle-derived cultured cells such as dermal papilla cells may be used to measure the activity of the keratinizing enzyme in the cells cultured in the presence of the hair-restoring agent.
In the present invention, the activity of keratinizing enzyme is measured for hair follicles or cultured cells derived from hair follicles of such experimental animals or humans, but the method is not particularly limited and is usually performed. It may be measured by the method.

For example, the skin of an experimental animal was collected, 1 mM of EDTA-containing 50 mM sodium dihydrogen phosphate aqueous solution was added thereto, and the homogenate obtained by homogenization was centrifuged at 27,000 G. , The enzymatic activity of SH oxidase contained in this, as is usually done, dithiothreitol (DTT)
With 7-fluoro-4-sulfamoyl-benzoxadiazole (ABD-F) to measure the fluorescence intensity, or with DTT and 5,5′-dithiobis-2-nitrobenzoic acid (DNTB). Examples thereof include a method of measuring the absorbance.

In the present invention, the activity value of the keratin enzyme thus measured is determined per weight of the sample (hair follicle, skin, etc.), or the skin is used when the skin is used as described above. The value per area amount is obtained, and the hair quality is evaluated using this value as an index. Further, by measuring the DNA amount and protein amount of the supernatant obtained in the above-mentioned experimental example, the activity value of keratinase per DNA (also referred to as the number of cells) or per protein can be obtained. Hair quality can also be evaluated as an index. That is, the larger the activity value per each evaluation unit (sample weight, skin area amount, cell number, protein, etc.) of the keratin enzyme obtained as described above, the thicker and stronger the hair is, and conversely If this value is small, it means that the hair is thin and the strength is low.

The method for evaluating a hair nourishing agent of the present invention utilizes this, and, for example, in an experimental animal or a human to which a hair nourishing agent has been administered and an experimental animal or a human to which no hair nourishing agent has been administered, or a hair nourishing agent administration site of the same experimental animal or human And the non-administered site, the activity of the keratinase per evaluation unit is compared to evaluate the hair nourishing effect of the hair nourishing agent. Further, since the method for evaluating a hair nourishing agent of the present invention can be carried out using human hair follicle cells, the keratinizing enzyme activity measurement of hair follicle cells of a hair nourishing agent user is performed regularly while using the hair nourishing agent. Specifically, it is possible to quantitatively evaluate whether or not the hair nourishing agent used by the user is actually effective.

In the present invention, the hair nourishing effect of the hair nourishing agent is evaluated as described above, and the results of experiments supporting the evaluation method of the present invention will be described below. The following evaluations were carried out on hair using 10 people each suffering from thinning hair and 10 people with normal hair as subjects, and the results were compared between the two.

First, the appearance of the hair and hair follicles of each test subject was observed under an optical microscope. In addition, the ease of hair removal was evaluated by measuring the number of hairs removed per day.
The cutting strength of hair was measured using Tensilon. On the other hand, the keratase activity of hair follicles was measured by the following method.

Hair was removed from each of the above-mentioned subjects and the roots of the hairs were collected.
TA-containing 50 mM sodium phosphate buffer (pH
7.6, hereinafter simply referred to as buffer), and homogenized. 27,000 G of each homogenate
Centrifugation was performed and the resulting supernatant was used as a sample for measuring keratin enzyme activity.

40 μL of 50 μL of the sample thus obtained
Specimen 1 was prepared by adding 25 μL of a buffer containing micromolar DTT and 175 μL of the buffer. Further, 200 μL of buffer was added to 50 μL of the sample to give a sample 2.

Immediately after preparing the sample 1, 100 μm
L was sampled and 0.1 mol sodium borate buffer (pH pH containing 2 mmol EDTA in advance).
(8.0) 900 μL was transferred to a tube, 1 mL of 0.1 mol borate buffer (pH 8.0) containing 100 μmol ABD-F was added thereto, and the mixture was treated at 50 ° C. for 30 minutes and then cooled with water. Furthermore, 0.1N
Hydrochloric acid (600 μL) was added, and the absorbance FI1 at 412 nm was measured. Further, the sample 2 was treated in the same manner, and the absorbance FI2 was measured.

Immediately after preparation of the sample 1, 100 μ
L was sampled and incubated at 37 ° C. for 2 hours, then treated as described above, and the absorbance FI at 412 nm was measured.
3 was measured. In addition, immediately after the preparation of the sample 2, 100 μL was sampled, incubated at 37 ° C. for 2 hours, and then similarly processed to measure the absorbance FI4.

From these measured absorbance values, the keratin enzyme activity value FI5 of the sample was determined using the following formula.

[0029]

## EQU1 ## FI5 = FI1-FI3- (FI2-FI4)

Further, regarding the supernatant obtained above, PI
Using the protein assay reagent kit from ERCE, the protein content of each supernatant was measured according to a standard method using bovine serum albumin as a standard, and the value obtained by dividing the above keratinase activity value by this protein content was evaluated. Used for.

Table 1 shows the results obtained in each of the above evaluation experiments.

[0032]

[Table 1]

As is clear from these results, compared to ordinary human hair and hair follicles, thin-haired human hair tends to be thin, easy to come off, and have low cutting strength, and the hair follicles are small. I understood. And, the keratin enzyme activity in the hair follicles of thin-haired people is at a lower level than the keratin enzyme activity in normal human hair follicles, and the observation of the actual hair condition,
It agrees with the measurement result. From this, it is clear that the hair condition can be known by measuring the enzyme activity of the hair follicle keratinase, and thus the hair nourishing effect can be evaluated.

[0034]

The keratin enzyme used in the method for evaluating the hair nourishing agent of the present invention is widely present mainly in the epidermis of hair and skin, and is involved in the synthesis of keratin and the like, and cuticles and cortex of hair, and further the horny layer of skin. Promotes biosynthesis. When a large amount of these enzymes are secreted or their activity is promoted, keratinization progresses to form an elastic stratum corneum in the skin, and keratin synthesis is promoted in hair to form cuticles and cortex. Resulting in the formation of thick, firm and tough hair.

Further, considering the hair and its surrounding tissues, the hair is at its root, the so-called hair follicle,
The cuticle of the hair and the hair follicle epithelial tissue are engaged with each other in a wedge shape to have a structure for fixing the hair. Therefore, as the thickness of the hair increases, the fixation of this hair becomes stronger and, as a result, it becomes more difficult for the hair to come off.

In other words, by measuring the activity of keratinizing enzyme in hair follicles, it is possible to understand the condition of thickening of hair, and the difficulty of pulling out the hair (ease). Is that you can know.

The evaluation method of the hair nourishing agent of the present invention utilizes the above-mentioned principle, and according to the evaluation of the hair nourishing agent of the present invention,
It is possible to appropriately evaluate the actual hair-growth effect, that is, the change in volume feeling.

Since the method for evaluating a hair nourishing agent of the present invention can be carried out using human hair follicle cells, if this is applied, the user of the hair nourishing agent can actually use the hair nourishing agent. It is possible to regularly and quantitatively evaluate the effect of the hair nourishing agent.

[0039]

EXAMPLES Examples of the present invention will be described below. Bamboo leaves, aloe extracts, minoxidil, carrots, and silkworms, which have been widely used as hair restorers, have been evaluated by the evaluation method of the present invention. Further, in order to confirm whether or not the evaluation method of the present invention is appropriate, carrot and silkworm were also evaluated for their hair loss-suppressing action and evaluation by a practical use test.

In the experiment, dried leaves of bamboo leaves and aloe extracts were extracted, and carrots and silkworms were extracted.
The extract liquid extracted with about 10 times the amount of 50% ethanol aqueous solution was used as it was.

(1) Measurement of keratinase activity value C3H mice (male, body weight 25-35, 5 groups of 5 mice each)
After removing the hair from the back of g), the hair removal skin of 5 groups of mice contains bamboo leaves, aloe extract, and minoxidil at the respective concentrations shown in Table 1, which are known to have a hair growth promoting action. 70% aqueous ethanol solution, carrot extract (L
A-P), silkworm extract, and 70% ethanol aqueous solution as a control on the depilated skin of the remaining 1 group of mice, respectively, 40 μL per animal, once a day, 5 days a week It was administered for a week.

Six hours after the final administration, the skin of the treated site was collected from each group of mice, and each of them was added to 9 volumes of 1 mM EDTA-containing 50 mM sodium dihydrogen phosphate aqueous solution. , Homogenized. This homogenate was centrifuged at 27,000 G, and the supernatant obtained was used to measure the keratinase activity value of each of the above hair nourishing agent administration groups and control group by the following method.

0.4 mL of the supernatant obtained above contained 0.1 mL of 2 mM DTT aqueous solution and 1 mM EDTA in 50 mM phosphate buffer (pH 7.6,
Hereinafter referred to simply as a buffer. ) 0.7 mL was added to make Sample 1. In addition, 0.1 mL of 2 mmol DTT aqueous solution
To the sample 2, 1.1 mL of buffer was added.

Immediately after preparing the sample 1, 0.3 mL was sampled and transferred to a tube to which 3 mL of a buffer containing 0.17 mmol of DTNB was added in advance.
The absorbance A1 at 2 nm was measured. Further, the sample 2 was treated in the same manner, and the absorbance A2 was measured.

The rest of the sample 1 was incubated at 37 ° C. for 30 minutes, 0.3 mL of which was sampled, and buffer 3 containing 0.17 mmol of DTNB in advance.
It was transferred to a tube to which mL was added, and the absorbance A3 at 412 nm was measured. In addition, the rest of sample 2 was
After incubating for 0 minutes, the same treatment is performed to obtain the absorbance A.
4 was measured.

Absorbances A1 to A4 thus measured
From the value of A5, A5 is calculated using the following formula, and the amount of oxidized DTT is calculated using the molar absorptivity of A5 and DTNB (13000 / mole · cm). ).

[0047]

## EQU00002 ## A5 = A1-A3- (A2-A4)

The amount of DNA in each of the supernatants obtained above was measured by a conventional method using a DNA amount measuring reagent Hoechst 33258 manufactured by Hoechst Co., Ltd. using calf thoracic line DNA as a standard. .

The evaluation was carried out by dividing the keratin enzyme activity value (Ac) obtained by the above-mentioned method by the above-mentioned DNA amount for each of the above-mentioned hair nourishing agent administration groups, and keratin enzyme activity value per unit DNA amount (AcD, The average value of 5 animals was calculated, and this was divided by the keratin enzyme activity value per unit amount of DNA (average value of 5 animals) of the control group obtained in the same manner, and this was multiplied by 100 (AeD). It was done using. In addition, AcD is a value showing the keratinase activity per number of cells.

Furthermore, regarding the supernatant obtained above, PI
Using a protein assay reagent kit from ERCE, the protein content of each supernatant was measured by a conventional method using bovine serum albumin as a standard, and the keratinase activity value (A
c) is divided by this, and the keratin enzyme activity value per protein (Ac
The average value of P5 animals) was divided by the keratin enzyme activity value per protein of the control group (average value of 5 animals) obtained in the same manner, and this was multiplied by 100 to obtain a value (AeP),
It was used for evaluation together with AeD. Table 2 shows the results.

[0051]

[Table 2]

Next, the hair loss inhibiting effect was evaluated for the carrot extract (LA-P) and the silkworm sperm extract among the above-mentioned hair nourishing agents using mice with rough hair.

(2) Measurement of Hair Loss Inhibitory Action First, a preliminary investigation was conducted to determine the course of coarse hair formation in 3 mice (male) with hair baldness, and the time to administer the hair-restoring agent was determined. The results of the investigation are shown in FIG. 1, and it was found that the coarsening of the hair in the mouse with porphyria markedly progressed up to 9 weeks of age, but was stabilized thereafter. Therefore, the administration period of the hair nourishing agent was set to 4 weeks from 5 weeks old to 9 weeks old.

Carrot extract (LA-P) was applied to the dorsal skin of 1 group of 3 groups of 5 week old mice (male) with 3 groups of 4 mice each.
, Silkworm extract on the back skin of the other 1 group, and 50% ethanol aqueous solution as a control on the back skin of the remaining 1 group, 100 μL per day, 5 days a week for 4 weeks It was administered continuously.

On the day after the end of administration, the hair of the administration area was shaved, the skin of the area was biopsied, and an arbitrary 12 points on the skin surface were photographed with a micro video (objective lens × 200) to measure the number of hairs. Was measured. The results are shown in FIG. 2 as an average value of 4 animals at 12 locations. In the figure, * indicates P <0.01 and + indicates P
<0.05 is represented respectively.

Further, the carrot extract (LA-P) and the silkworm sperm extract among the above-mentioned hair nourishing agents were evaluated for their hair growth promoting action.

(3) Practical use test Twelve female panelists having trouble with firmness and stiffness of hair were divided into groups of six, and 16 hairs were sampled from a certain area near the top of the head. 1
For 0 hairs, a cross-sectional area of about 5 mm from the root of the hair and a long diameter were measured, and then tensile strength was measured to obtain a cross-sectional area and a value per long diameter as an average value of 10 hairs. For the remaining 6 fibers, a cross-sectional area and major axis of about 5 mm from the root of the hair were measured in the same manner as described above, and then viscoelasticity was measured to obtain a cross-sectional area and a value per major diameter as an average value of 6 fibers.

Then, the above carrot extract (LA-P)
And 20% by weight of each of the silkworm sperm extract in a 50% ethanol aqueous solution containing 3% of 1,3-butylene glycol, were applied to each group of panelists in the same manner as a normal hair restorer for 1 day. I got it to be used continuously for 2 months. Two months after the start of use, hair was sampled in the same manner as above, and the cross-sectional area, the tensile strength per major axis and the cross-sectional area, and the viscoelasticity per major axis were measured.

For the evaluation, the percentage of the value after using the sample to the value before using was used. Table 3 shows the average of 6 panelists with standard deviation. In the table, * is P <0.01
And + means that P <0.05.

[0060]

[Table 3]

These test results show that bamboo leaves, aloe extract, minoxidil, which are considered to have an excellent hair-growth effect in the evaluation of conventional hair-growth agents, that is, in the evaluation result of the hair growth promoting action,
Even in carrots and silkworms, in the evaluation results of the keratinizing enzyme promoting action used in the method for evaluating a hair nourishing agent of the present invention, aloe extract, minoxidil, carrots and silkworms are excellent in hair-feeding action,
It shows that bamboo leaves do not have excellent hair-feeding effect by suppressing keratinizing enzyme. This is in good agreement with the fact that bamboo leaves are generally said to have a poorer actual hair-growth effect than other hair-growth agents.

Further, regarding carrots and silkworms determined to have an excellent hair-growth effect by the method for evaluating a hair nourishing agent of the present invention, hair loss suppression and hair quality are evaluated based on the results of evaluation of hair loss suppressing effect and results of actual use test. It was confirmed that the actual hair nourishing effect such as improvement was excellent.

From the above, it is clear that the method for evaluating a hair nourishing agent of the present invention using the activity value of keratinizing enzyme as an index can appropriately evaluate the actual hair nourishing effect.

[0064]

According to the method of evaluating a hair nourishing agent of the present invention, it is possible to appropriately evaluate the actual hair nourishing effect.

[Brief description of drawings]

FIG. 1 is a graph showing the change over time in the number of remaining hairs in a mouse with osteoporosis.

FIG. 2 is a graph showing the number of remaining hairs in a mouse with hair trophoblastus to which various hair nourishing agents are applied

Claims (2)

[Claims] 1. A hair nourishing agent, characterized in that the hair nourishing effect of the hair nourishing agent is evaluated by measuring the activity of a keratinizing enzyme in hair follicles or cultured cells derived from hair follicles of experimental animals or humans to which the hair nourishing agent is administered. Evaluation method. 2. The method for evaluating a hair nourishing agent according to claim 1, wherein the keratinizing enzyme is sulfhydryl oxidase.